This invention relates to a method of reducing the level of cytokines and their precursors in mammals and to compounds and compositions useful therein.
In particular, the invention pertains to a class of compounds which inhibit the action of phosphodiesterases, particularly PDE III and PDE IV, and the formation of TNF.alpha. and NF.sub..kappa. B. In a first embodiment, the compounds of the present invention can be diagrammatically represented by the formula: ##STR1## in which: R.sup.5 is:
(i) the divalent residue of pyridine, pyrrolidine, imidizole, naphthalene, or thiophene, wherein the two bonds of the divalent residue are on vicinal ring carbon atoms; PA2 (ii) a divalent cycloalkyl of 4 to 10 carbon atoms, unsubstituted or substituted with one or more substituents each selected independently of the other from the group consisting of nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, substituted amino, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms, phenyl or halo; PA2 (iii) di-substituted vinylene, substituted with nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, carbamoyl substituted with and alkyl of 1 to 3 carbon atoms, acetoxy, carboxy, hydroxy, amino, amino substituted with an alkyl of 1 to 3 carbon atoms, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or halo; or PA2 (iv) ethylene, unsubstituted or substituted with 1 to 2 substituents each selected independently from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, carbamoyl substituted with and alkyl of 1 to 3 carbon atoms, acetoxy, carboxy, hydroxy, amino, amino, substituted with an alkyl of 1 to 3 carbon atoms, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or halo; PA2 (i) cyclic or bicyclic alkyl of 4 to 12 carbon atoms; PA2 (ii) pyridyl; PA2 (iii) phenyl substituted with one or more substituents each selected independently of the other from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, straight or branched alkyl of 1 to 10 carbon atoms, straight or branched alkoxy of 1 to 10 carbon atoms, or halo; PA2 (iv) benzyl substituted with one to three substituents each selected independently from the group consisting of nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 10 carbon atoms, or halo; PA2 (v) naphthyl; or PA2 (vi) benzyloxy; PA2 (i) the vicinally divalent residue of pyridine, pyrrolidine, imidizole, naphthalene, or thiophene, wherein the two bonds of the divalent residue are on vicinal ring carbon atoms; PA2 (ii) a vicinally divalent cycloalkyl of 4-10 carbon atoms, unsubstituted or substituted with 1 to 3 substituents each selected independently from the group consisting of nitro, cyano, halo, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, substituted amino, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms, phenyl; PA2 (iii) di-substituted vinylene, substituted with nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, carbamoyl substituted with and alkyl of 1 to 3 carbon atoms, acetoxy, carboxy, hydroxy, amino, amino substituted with an alkyl of 1 to 3 carbon atoms, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or halo; or PA2 (iv) ethylene, unsubstituted or substituted with 1 to 2 substituents each selected independently from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, carbamoyl substituted with and alkyl of 1 to 3 carbon atoms, acetoxy, carboxy, hydroxy, amino, amino substituted with an alkyl of 1 to 3 carbon atoms, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or halo;
R.sup.6 is --CO--, --CH.sub.2 --, --CH.sub.2 CO--, or --SO.sub.2 --; PA1 R.sup.7 is PA1 Y is --COX, --C.tbd.N, --OR.sup.8, alkyl of 1 to 5 carbon atoms, or aryl; PA1 X is --NH.sub.2, --OH, --NHR, --R.sup.9, --OR.sup.9, or alkyl of 1 to 5 carbon atoms; PA1 R.sup.8 is hydrogen or lower alkyl; PA1 R.sup.9 is alkyl or benzyl; and, PA1 n has a value of 0, 1, 2, or 3. PA1 R.sup.3 is monocycloalkyl of up to 10 carbon atoms, polycycloalkyl of up to 10 carbon atoms, or benzocyclic alkyl of up to 10 carbon atoms; PA1 X is --CH.sub.2 -- or --O--; PA1 R.sup.5 is: PA1 R.sup.6 is --CO--, --CH.sub.2 --, or --CH.sub.2 CO--; PA1 Y is --COX, --C.tbd.N, --OR.sup.8, alkyl of 1 to 5 carbon atoms, or aryl; PA1 X is --NH.sub.2, --OH, --NHR, --R.sup.9, --OR.sup.9, or alkyl of 1 to 5 carbon atoms; PA1 R.sup.8 is hydrogen or lower alkyl; PA1 R.sup.9 is alkyl or benzyl; and, PA1 n has a value of 0, 1, 2, or 3.
Within this group, Y is preferably --C.tbd.N or --CO(CH.sub.2).sub.m CH.sub.3 in which m has a value of 0, 1, 2, or 3; and
In a second embodiment, the compounds of the present invention can be diagrammatically represented by the formula: ##STR2## in which: one of R.sup.1 and R.sup.2 is R.sup.3 --X-- and the other is hydrogen, nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, lower alkyl, lower alkoxy, halo, or R.sup.3 --X--;
The term alkyl as used herein denotes a univalent saturated branched or straight hydrocarbon chain. Unless otherwise stated, such chains can contain from 1 to 18 carbon atoms. Representative of such alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, isohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, and the like. When qualified by "lower", the alkyl group will contain from 1 to 6 carbon atoms. The same carbon content applies to the parent term "alkane" and to derivative terms such as "alkoxy".
The term cycloalkyl as used herein denotes a univalent saturated cyclic hydrocarbon chain. Unless otherwise stated, such chains can contain up to 18 carbon atoms. Monocyclicalkyl refers to groups having a single ring group. Polycycloalkyl denotes hydrocarbon systems containing two or more ring systems with two or more ring carbon atoms in common. Benzocycloalkyl signifies a monocyclicalkyl group fused to a benzo group.
Representative of monocycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, cyclotridecyl, cyclotetradecyl, cyclopentadecyl, cyclohexadecyl, cycloheptadecyl, and cyclooctadecyl. Representative of polycycloalkyl include bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, and bicyclo[2.2.2]octyl. Benzocycloalkyl is typified by tetrahydronaphthyl, indanyl, and 1.2-benzocycloheptanyl.
Tumor necrosis factor .alpha., or TNF.alpha., is a cytokine which is released primarily by mono nuclear phagocytes in response to a number immunostimulators. When administered to animals or humans, it causes inflammation, fever, cardiovascular effects, hemorrhage, coagulation, and acute phase responses similar to those seen during acute infections and shock states. Excessive or unregulated TNF.alpha. production thus has been implicated in a number of disease conditions. These include endotoxemia and/or toxic shock syndrome {Tracey et al., Nature 330, 662-664 (1987) and Hinshaw et al., Circ. Shock 30, 279-292 (1990)}; cachexia {Dezube et al., Lancet, 335 (8690), 662 (1990)} and Adult Respiratory Distress Syndrome where TNF.alpha. concentration in excess of 12,000 pg/mL have been detected in pulmonary aspirates from ARDS patients {Millar et al., Lancet 2(8665), 712-714 (1989)}. Systemic infusion of recombinant TNF.alpha. also resulted in changes typically seen in ARDS {Ferrai-Baliviera et al., Arch. Surg. 124(12), 1400-1405 (1989)}.
TNF.alpha. appears to be involved in bone resorption diseases, including arthritis. When activated, leukocytes will produce bone-resorption, an activity to which the data suggest TNF.alpha. contributes. {Bertolini et al., Nature 319, 516-518 (1986) and Johnson et al, Endocrinology 124(3), 1424-1427 (1989).} TNF.alpha. also has been shown to stimulate bone resorption and inhibit bone formation in vitro and in vivo through stimulation of osteoclast formation and activation combined with inhibition of osteoblast function. Although TNF.alpha. may be involved in many bone resorption diseases, including arthritis, the most compelling link with disease is the association between production of TNF.alpha. by tumor or host tissues and malignancy associated hypercalcemia {Calci. Tissue Int. (US) 46(Suppl.), S3-10 (1990)}. In Graft versus Host Reaction, increased serum TNF.alpha. levels have been associated with major complication following acute allogenic bone marrow transplants {Holler et al, Blood, 75(4), 1011-1016(1990)}.
Cerebral malaria is a lethal hyperacute neurological syndrome associated with high blood levels of TNF.alpha. and the most severe complication occurring in malaria patients. Levels of serum TNF.alpha. correlated directly with the severity of disease and the prognosis in patients with acute malaria attacks {Grau et al., N. Engl. J Med. 320(24), 1586-1591 (1989)}.
Macrophage-induced angiogenesis TNF.alpha. is known to be mediated by TNF.alpha.. Leibovich et al. {Nature, 329, 630-632 (1987)} showed TNF.alpha. induces in vivo capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membranes at very low doses and suggest TNF.alpha. is a candidate for inducing angiogenesis in inflammation, wound repair, and tumor growth. TNF.alpha. production also has been associated with cancerous conditions, particularly induced tumors {Ching et al., Brit. J. Cancer, (1955) 72, 339-343, and Koch, Progress in Medicinal Chemistry, 22, 166-242 (1985)}.
TNF.alpha. also plays a role in the area of chronic pulmonary inflammatory diseases. The deposition of silica particles leads to silicosis, a disease of progressive respiratory failure caused by a fibrotic reaction. Antibody to TNF.alpha. completely blocked the silica-induced lung fibrosis in mice {Pignet et al, Nature, 344, 245-247 (1990)}. High levels of TNF.alpha. production (in the serum and in isolated macrophages) have been demonstrated in animal models of silica and asbestos induced fibrosis {Bissonnette et al., Inflammation 13(3), 329-339 (1989)}. Alveolar macrophages from pulmonary sarcoidosis patients have also been found to spontaneously release massive quantities of TNF.alpha. as compared with macrophages from normal donors {Baughman et al, J. Lab. Clin. Med. 115(1), 36-42 (1990)}.
TNF.alpha. is also implicated in the inflammatory response which follows reperfusion, called reperfusion injury, and is a major cause of tissue damage after loss of blood flow {Vedder et al., PNAS 87, 2643-2646 (1990)}. TNF.alpha. also alters the properties of endothelial cells and has various procoagulant activities, such as producing an increase in tissue factor pro-coagulant activity and suppression of the anticoagulant protein C pathway as well as down-regulating the expression of thrombomodulin {Sherry et al., J. Cell Biol. 107, 1269-1277 (1988)}. TNF.alpha. has pro-inflammatory activities which together with its early production (during the initial stage of an inflammatory event) make it a likely mediator of tissue injury in several important disorders including but not limited to, myocardial infarction, stroke and circulatory shock. Of specific importance may be TNF.alpha.-induced expression of adhesion molecules, such as intercellular adhesion molecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) on endothelial cells {Munro et al., Am. J. Path. 135(1), 121-132 (1989)}.
TNF.alpha. blockage with monoclonal anti-TNF.alpha. antibodies has been shown to be beneficial in rheumatoid arthritis {Elliot et al., Int. J. Pharmac. 1995 17(2), 141-145}. High levels of TNF.alpha. are associated with Crohn's disease {von Dullemen et al., Gastroenterology, 1995 109(1), 129-135} and clinical benefit has been achieved with TNF.alpha. antibody treatment.
Moreover, it now is known that TNF.alpha. is a potent activator of retrovirus replication including activation of HIV-1. {Duh et al., Proc. Nat. Acad. Sci. 86, 5974-5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87, 782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J. Immunol. 142, 431-438 (1989); Poll et al., AIDS Res. Hum. Retrovirus, 191-197 (1992)}. AIDS results from the infection of T lymphocytes with Human Immunodeficiency Virus (HIV). At least three types or strains of HIV have been identified, i.e., HIV-1, HIV-2 and HIV-3. As a consequence of HIV infection, T-cell mediated immunity is impaired and infected individuals manifest severe opportunistic infections and/or unusual neoplasms. HIV entry into the T lymphocyte requires T lymphocyte activation. Other viruses, such as HIV-1, HIV-2 infect T lymphocytes after T cell activation and such virus protein expression and/or replication is mediated or maintained by such T cell activation. Once an activated T lymphocyte is infected with HIV, the T lymphocyte must continue to be maintained in an activated state to permit HIV gene expression and/or HIV replication. Cytokines, specifically TNF.alpha., are implicated in activated T-cell mediated HIV protein expression and/or virus replication by playing a role in maintaining T lymphocyte activation. Therefore, interference with cytokine activity such as by prevention or inhibition of cytokine production, notably TNF.alpha., in an HIV-infected individual assists in limiting the maintenance of T lymphocyte caused by HIV infection.
Monocytes, macrophages, and related cells, such as kupffer and glial cells, also have been implicated in maintenance of the HIV infection. These cells, like T cells, are targets for viral replication and the level of viral replication is dependent upon the activation state of the cells. {Rosenberg et al., The Immunopathogenesis of HIV Infection, Advances in Immunology, 57 (1989)}. Cytokines, such as TNF.alpha., have been shown to activate HIV replication in monocytes and/or macrophages {Poli et al., Proc. Natl. Acad. Sci., 87, 782-784 (1990)}, therefore, prevention or inhibition of cytokine production or activity aids in limiting HIV progression for T cells. Additional studies have identified TNF.alpha. as a common factor in the activation of HIV in vitro and has provided a clear mechanism of action via a nuclear regulatory protein found in the cytoplasm of cells (Osborn, et al., PNAS 86 2336-2340). This evidence suggests that a reduction of TNF.alpha. synthesis may have an antiviral effect in HIV infections, by reducing the transcription and thus virus production.
AIDS viral replication of latent HIV in T cell and macrophage lines can be induced by TNF.alpha. {Folks et al., PNAS 86, 2365-2368 (1989)}. A molecular mechanism for the virus inducing activity is suggested by TNF.alpha.'s ability to activate a gene regulatory protein (NF.kappa.B) found in the cytoplasm of cells, which promotes HIV replication through binding to a viral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 2336-2340 (1989)}. TNF.alpha. in AIDS associated cachexia is suggested by elevated serum TNF.alpha. and high levels of spontaneous TNF.alpha. production in peripheral blood monocytes from patients {Wright et al., J. Immunol. 141(1), 99-104 (1988)}. TNF.alpha. has been implicated in various roles with other viral infections, such as the cytomegalia virus (CMV), influenza virus, adenovirus, and the herpes family of viruses for similar reasons as those noted.
The nuclear factor .kappa.B (NF.kappa.B) is a pleiotropic transcriptional activator (Lenardo, et al., Cell 1989, 58, 227-29). NF.kappa.B has been implicated as a transcriptional activator in a variety of disease and inflammatory states and is thought to regulate cytokine levels including but not limited to TNF.alpha. and also to be an activator of HIV transcription (Dbaibo, et al., J. Biol. Chem. 1993, 17762-66; Duh et al., Proc. Natl. Acad. Sci. 1989, 86, 5974-78; Bachelerie et al., Nature 1991, 350, 709-12; Boswas et al., J. Acquired Immune Deficiency Syndrome 1993, 6, 778-786; Suzuki et al., Biochem. And Biophys. Res. Comm. 1993, 193, 277-83; Suzuki et al., Biochem. And Biophys. Res Comm. 1992, 189, 1709-15; Suzuki et al., Biochem. Mol. Bio. Int. 1993, 31(4), 693-700; Shakhov et al., Proc. Natl. Acad. Sci. USA 1990, 171, 35-47; and Staal et al., Proc. Natl. Acad. Sci. USA 1990, 87, 9943-47). Thus, inhibition of NF.kappa.B binding can regulate transcription of cytokine gene(s) and through this modulation and other mechanisms be useful in the inhibition of a multitude of disease states. The compounds described herein can inhibit the action of NK.kappa.B in the nucleus and thus are useful in the treatment of a variety of diseases including but not limited to rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, septic shock, septis, endotoxic shock, graft versus host disease, wasting, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV, AIDS, and opportunistic infections in AIDS. TNF.alpha. and NK.kappa.B levels are influenced by a reciprocal feedback loop. As noted above, the compounds of the present invention affect the levels of both TNF.alpha. and NK.kappa.B.
Many cellular functions are mediated by levels of adenosine 3',5'-cyclic monophosphate (cAMP). Such cellular functions can contribute to inflammatory conditions and diseases including asthma, inflammation, and other conditions (Lowe and Cheng, Drugs of the Future, 17(9), 799-807, 1992). It has been shown that the elevation of cAMP in inflammatory leukocytes inhibits their activation and the subsequent release of inflammatory mediators, including TNF.alpha. and NK.kappa.B. Increased levels of cAMP also leads to the relaxation of airway smooth muscle. Phosphodiesterases control the level of cAMP through hydrolysis and inhibitors of phosphodiesterases have been shown to increase cAMP levels.
Decreasing TNF.alpha. levels and/or increasing cAMP levels thus constitutes a valuable therapeutic strategy for the treatment of many inflammatory, infectious, immunological or malignant diseases. These include but are not restricted to septic shock, sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, post ischemic reperfusion injury, malaria, mycobacterial infection, meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, opportunistic infections in AIDS, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury. Prior efforts directed to the suppression of the effects of TNF.alpha. have ranged from the utilization of steroids such as dexamethasone and prednisolone to the use of both polyclonal and monoclonal antibodies {Beutler et al., Science 234, 470-474 (1985); WO 92/11383}.
The compounds claimed in this patent inhibit the action of NK.kappa.B in the nucleus and thus are useful in the treatment of a variety of diseases including but not limited to rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, septic shock, septis, endotoxic shock, graft versus host disease, wasting, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV, AIDS, and opportunistic infections in AIDS.
It is not known at this time, however, how the compounds of the present invention regulate the levels of TNF.alpha., NK.kappa.B, or both. As noted above, the compounds of the present invention affect the levels of both TNF.alpha. and NF.kappa.B.
The compounds can be used, under the supervision of qualified professionals, to inhibit the undesirable effects of TNF.alpha. or phosphodiesterase. The compounds can be administered orally, rectally, or parenterally, alone or in combination with other therapeutic agents including antibiotics, steroids, etc., to a mammal in need of treatment. Oral dosage forms include tablets, capsules, dragees, and similar shaped, compressed pharmaceutical forms. Isotonic saline solutions containing 20-100 milligrams/milliliter can be used for parenteral administration which includes intramuscular, intrathecal, intravenous and intra-arterial routes of administration. Rectal administration can be effected through the use of suppositories formulated from conventional carriers such as cocoa butter.
Dosage regimens must be titrated to the particular indication, the age, weight, and general physical condition of the patient, and the response desired but generally doses will be from about 1 to about 1000 milligrams/day as needed in single or multiple daily administration. In general, an initial treatment regimen can be copied from that known to be effective in interfering with TNF.alpha. activity for other TNF.alpha. mediated disease states by the compounds of the present invention. Treated individuals will be regularly checked for T cell numbers and T4/T8 ratios and/or measures of viremia such as levels of reverse transcriptase or viral proteins, and/or for progression of cytokine-mediated disease associated problems such as cachexia or muscle degeneration. If no effect is observed following the normal treatment regimen, then the amount of cytokine activity interfering agent administered is increased, e.g., by fifty percent a week.
The compounds of the present invention can also be used topically in the treatment or prophylaxis of topical disease states mediated or exacerbated by excessive TNF.alpha. production, such as viral infections, for example those caused by the herpes viruses or viral conjunctivitis, psoriasis, other skin disorders and diseases, etc.
The compounds can also be used in the veterinary treatment of mammals other than humans in need of prevention or inhibition of TNFA production. TNF.alpha. mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted above, but in particular viral infections. Examples include feline immunodeficiency virus, equine infectious anaemia virus, caprine arthritis virus, visna virus, and maedi virus, as well as other lentiviruses.
These compounds possess at least one center of chirality and thus will exist as optical isomers. Both the racemates of these isomers and the individual isomers themselves, as well as diastereoisomers when there are two or more chiral centers, are within the scope of the present invention. The racemates can be used as such or can be separated into their individual isomers mechanically as by chromatography using a chiral absorbent. Alternatively, the individual isomers can be prepared in chiral form or separated chemically from a mixture by forming salts with a chiral acid, such as the individual enantiomers of 10-camphorsulfonic acid, camphoric acid, alpha-bromocamphoric acid, methoxyacetic acid, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, and the like, and then freeing one or both of the resolved bases, optionally repeating the process, so as to obtain either or both isomers substantially free of the other; i.e., in a form having an optical purity of &gt;95%.
Prevention or inhibition of production of TNF.alpha. by these compounds can be conveniently assayed using methods known in the art. For example, TNF.alpha. Inhibition Assays can be performed as follows:
PBMC isolation: PBMC from normal donors were obtained by Ficoll-Hypaque density centrifugation. Cells were cultured in RPMI supplemented with 10% AB+ serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin.
PBMC suspensions: Drugs were dissolved in DMSO (Sigma Chemical), further dilutions were done in supplemented RPMI. The final DMSO concentration in the presence or absence of drug in the PBMC suspensions was 0.25 wt %. Drugs were assayed at half-log dilutions starting at 50 mg/mL. Drugs were added to PBMC (10.sup.6 cells/mL) in 96 wells plates one hour before the addition of LPS.
Cell stimulation: PBMC (10.sup.6 cells/mL) in the presence or absence of drug were stimulated by treatment with 1 mg/nL of LPS from Salmonella minnesota R595 (List Biological Labs, Campbell, Calif.). Cells were then incubated at 37.degree. C. for 18-20 hours. Supernatants were then harvested and assayed immediately for TNF.alpha. levels or kept frozen at -70.degree. C. (for not more than 4 days) until assayed.
TNF.alpha. Determination: The concentration of TNF.alpha. in the supernatant was determined by human TNF.alpha. ELISA kits (ENDOGEN, Boston, Mass.) according to the manufacturer's directions.
Another assay procedure utilizes plates (Nunc Immunoplates, Roskilde, DK) which are treated with 5 mg/mL of purified rabbit anti-TNF.alpha. antibodies at 4.degree. C. for 12 to 14 hours. The plates then are blocked for 2 hours at 25.degree. C. with PBS/0.05% Tween containing 5 milligrams/millilter BSA. After washing, 100 mL of unknowns as well as controls are applied and the plates incubated at 4.degree. C. for 12 to 14 hours. The plates are washed and assayed with a conjugate of peroxidase (horseradish) and mouse anti-TNF.alpha. monoclonal antibodies, and the color developed with o-phenylenediamine in phosphate citrate buffer containing 0.012% hydrogen peroxide and read at 492 nm.
The compounds can be prepared using methods which are known per se. for example, a cyclic anhydride of lactone can be reacted with the appropriate amine: ##STR3## in which R.sup.5, R.sup.6, R.sup.7, Y, and n are as defined above. The reaction can be effected analogously to the methods described in U.K. Patent Specification No. 1,036,694, the disclosure of which is incorporated herein by reference. Optionally acetic acid, with or without sodium acetate, can be added.
In place of the acid anhydride or lactone, one can utilize an N-carbethoxy derivative of the formula: ##STR4##
In a further embodiment, compounds in which R.sup.6 is --CH.sub.2 -- can be formed through condensation of a dialdehyde with a disubstituted aromatic compound in the presence of refluxing acetic acid utilizing the method of Griggs et al., J.Chem. Soc. Chem. Comm., 1985, 1183-1184, the disclosure of which is incorporated herein by reference.
The disubstituted aromatic starting materials can be obtained through condensation of an appropriately substituted aldehyde and malonic acid, with intermediate formation of the amidine and subsequent decarboxylation.
The disubstituted aldehydes can be prepared utilizing classical methods for ether formation; e.g., reaction with the appropriate bromide in the presence of potassium carbonate. Numerous cycloalkyloxy benzaldehydes and procedures for preparing them are described in the literature. See, e.g., Ashton et al., J. Med. Chem., 1994, 37, 1696-1703; Saccomano et al., J. Med. Chem., 1994, 34,, 291-298; and Cheng et al., Org. and Med. Chem. Lett., 1995, 5(17), 1969-1972, the disclosures of which are incorporated herein by reference.
Typical compounds include 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl)propion ic acid, 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3-ethoxy-4-cyclohexyloxyphenyl)propionic acid, 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3-methoxy-4-ethoxyphenyl)propionic acid, 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3, 4-dimethoxyphenyl)propionic acid, 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)propioni c acid, 3-(1-oxo-4-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propionic acid, 3-(1-oxo-5-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propionic acid, 3-(1-oxo-4-azaisoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)propionic acid, 3-(1-oxo-4-azaisoindol-2-yl)-3-(3-methoxy-4-ethoxyphenyl)propionic acid, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-cyclopentyloxy-4-cyclohexyloxyphen yl)propionamide, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-ethoxy-4-cyclohexyloxyphenyl)-prop ionamide, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-methoxy-4-cyclobutyloxyphenyl)prop ionamide, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)pro pionamide, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)pro pionamide, 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propi onamide, 3-(1,3-dioxo-5-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propi onamide, 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)propio namide, 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-methoxy-4-cyclobutyloxyphenyl)propio namide, 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3-cyclopentyloxy-4-cyclohexyloxyphenyl)p ropionic acid, 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl)propion amide, 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3-ethoxy-4-cyclohexyloxyphenyl)propionam ide, 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyl)propionamide , 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3,4-dicyclopentyloxyphenyl)propionamide , 3-(1-oxobenzo[f]isoindol-2-yl)-3-(3,4-dicyclohexyloxyphenyl)propionamide, 3-(1-oxo-4-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propionam ide, 3-(1-oxo-5-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propionam ide, 3-(1-oxo-4-azaisoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)propionami de, 3-(1-oxo-4-azaisoindol-2-yl)-3-(3-cyclohexyloxy-4-ethoxyphenyl)propionamid e,3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl)pr opionic acid, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-ethoxy-4-cyclohexyloxyphenyl)-prop ionic acid, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-methoxy-4-cyclohexyloxyphenyl)-pro pionic acid, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3,4-dicyclohexyloxyphenyl)propionic acid, 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3, 4-dicyclopentyloxyphenyl)propionic acid, 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propi onic acid, 3-(1,3-dioxo-5-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propi onic acid, 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)propio nic acid, 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)propio nic acid, methyl 3-(1,3-dioxobenzo[flisoindol-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl)pro pionate, methyl 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-ethoxy-4-cyclohexyloxyphenyl)propi onate, methyl 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)prop ionate, methyl 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3,4-dicyclopentyloxyphenyl)propionat e, methyl 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3,4-dicyclohexyloxyphenyl)propionate , methyl 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propi onate, methyl 3-(1,3-dioxo-5-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propi onate, methyl 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)propio nate, ethyl 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-pr opionate, ethyl 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-ethoxy-4-cyclohexyloxyphenyl)propi onate, ethyl 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3-methoxy-4-ethoxyphenyl)propionate, ethyl 3-(1,3-dioxobenzo[f]isoindol-2-yl)-3-(3, 4-dimethoxyphenyl)propionate, ethyl 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propi onate, ethyl 3-(1,3-dioxo-5-azaisoindol-2-yl)-3-(3-methoxy-4-cyclopentyloxyphenyl)propi onate, ethyl and 3-(1,3-dioxo-4-azaisoindol-2-yl)-3-(3-ethoxy-4-cyclopentyloxyphenyl)propio nate.
Representative aldehyde starting materials include 3-cyclopentyloxy4-methoxybenzaldehyde, 3-cyclopentyloxy-4-ethoxybenzaldehyde, 3-cyclohexyloxy-4-cyclohexyloxybenzaldehyde, 3-(exo-bicyclo[2.2.1]hept2-yloxy)-4-methoxybenzaldehyde, 3-(endo-bicyclo[2.2.1]hept-2-yloxy)-4-methoxybenzaldehyde, 3-(bicyclo[2.2.2]oct-2-yloxy)-4-methoxybenzaldehyde, 3-(bicyclo-[3.2.1]oct-2-yloxy)-4-methoxybenzaldehyde, 3-indan-2-yloxy-4-methoxybenzaldehyde, and 3-(endo-benzobicyclo(2.2.1]hept-2-yloxy)-4-methoxybenzaldehyde.